Cells must merge membranes in order to mix aqueous spaces. We have made progress this year in developing new methods for the characterization of fusion pores, and the identification and characterization of eukaryotic fusion proteins. Using double whole-cell recording technique. junctional conductance was measured during baculovirus-mediated syncytia formation of insect cells. Fusion was triggered by low pH. Symmetrical currents in each cell of the pair measured the fusion pore conductance directly. It started as abrupt (0.1-0.2 ms rise-time) pore formation with a distribution of conductances centered at l nS.- widened slowly in the time scale of tens of seconds and then increased abruptly. Our previous method, time-resolved admittance measurement, was performed to compare two methods: both gave similar results. Thus our previous methods of measuring fusion pores was confirmed. The new method will allow us to improve the time resolution of the fusion pore conductance by 1-2 orders of magnitude. A multiple- frequency, digital lock-in amplifier was implemented on a microcomputer to extend our measurements of fusion pores so as to calculate the fusion pore conductance more precisely in the presence of noise. To determine the proteins involved in fusion pore formation, thiol group sensitivity was studied. Amino-dextran derivatives of the reversible thiol reagent SPDP inhibit exocytosis inversely with size. This implies that the susceptible thiol groups are not buried deeply but are in a relatively exposed hydrophilic environment. Thiol-reactive proteins are involved in triggering fusion rather than downstream events, since no fusion is seen upon the removal of the SPDP block by DTT treatment. GTP-triggered fusion of rat liver microsomes was strongly inhibited by sodium metaperiodate. The restorative effect of DTT indicates that at least the reversible portion of the inhibition is caused by oxidation of protein vicinal thiol groups with the formation of disulfide bonds. Selective labeling of periodate-sensitive thiols revealed one major labeled polypeptide with M.W. of 15 kDa. The correlation between oxidation and reduction of the protein and inhibition and restoration of the microsomal fusion, respectively, suggests that this protein may be part of the GTP-dependent microsomal fusion machinery.
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